PROJECT SUMMARY Changes in the glycosylation pattern of cell surface proteins and lipids are increasingly being recognized as an important factor in controlling cellular activity. There is growing interest in how these changes can be regulated to affect physiologic processes in health and disease. The goal of this proposal is to understand how a specific carbohydrate modification on the surface of cells of the immune system influences the function of these cells during states of inflammation and infection. As a key member of cellular innate and adaptive immune responses, monocytes are recruited to pulmonary sites of infection where they differentiate into macrophages and dendritic cells before migrating through the lymphatic system. This targeted migration and programmed maturation of monocytes and monocyte-derived cells is guided by specific, highly-organized cell-cell and receptor-ligand interactions, many of which are modulated by glycosylation of cell surface receptors and adhesion molecules. Polysialic acid (polySia) is a unique glycan modification of at least three such proteins, neural cell adhesion molecule (NCAM/CD56), neuropilin-2 (NRP-2), and E-selectin ligand-1 (ESL-1), that are expressed at different stages of monocyte maturation. We will test the hypothesis that regulated expression of polySia on monocytes as they differentiate into macrophages and dendritic cells, and on neutrophils that also are polysialylated, helps direct cell homing and a well-orchestrated immune response during pulmonary infection with viral and bacterial pathogens. Mice that are deficient in expression of the enzyme that synthesizes polySia in leukocytes (ST8 SiaIV-/-) and of the carrier proteins (NCAM/CD56-/- and NRP-2-/-) will be used for in vitro and in vivo studies. Our Specific Aims will i) establish the impact in vivo of polySia on monocytes, macrophages, DC and neutrophils in the targeted immune response in a murine model of bacterial and viral pneumonia; ii) define the mechanism(s) through which polysialylated proteins on the surface of human and murine monocytes, macrophages, DC and neutrophils control adhesion to and migration across pulmonary microvascular monolayers; and iii) define and analyze the regulated expression of polySia and of polysialylated proteins during differentiation of primary monocytes into DC and macrophages. We expect to identify specific cell-cell and receptor-ligand interactions that polySia promotes or interferes with during different stages of the monocyte/macrophage/dendritic cell maturation and migratory processes, as well during neutrophil recruitment. The results from our studies will provide a blueprint to engineer levels of expression of polySia and/or carrier proteins in myeloid cells in order to optimize migration to and from sites of infection/inflammation to improve the overall immune response.